The use of antibiotics to treat bacterial infections has resulted in the development of drug resistance. Drug resistant infections are responsible for over 11,000 deaths annually in the US and cost twice as much to treat as their susceptible counterparts. New therapeutic strategies to combat bacterial infections are needed. One promising approach is to target bacterial virulence, which would reduce pathogenicity and facilitate clearance of the infection by the host. Hyaluronidase is an enzyme secreted by pathogens including Staphylococcus aureus that is responsible for facilitating growth and penetration, and is therefore referred to as a spreading factor. There are currently no known inhibitors of S. aureus hyaluronidase. Our preliminary data show that fungi isolated from the botanical Anemopsis californica possess anti-hyaluronidase activity. The first goal of this project is to identify compounds from these active fungal extracts that are responsible for the observed activity. We will be using a mass spectrometry based assay developed by our laboratory that monitors hyaluronidase inhibition to guide isolation. Isolated compounds will be screened for broad-spectrum anti- hyaluronidase activity against both S. aureus and Streptococcus agalactiae, as well as for antimicrobial activity. The second goal of this proposal is to develop a untargeted synergy-directed fractionation approach that allows for the identification of combinations of compounds with anti-hyaluronidase activity. Our preliminary data suggest that multiple components are responsible for the observed activity of the fungal extracts, and the approach developed in this proposal will be employed to identify these. Numerous literature reports suggest that the biological activity of natural product extracts can result from the combined, often synergistic, activities of multiple compounds. Thus, the new strategies developed herein may be applicable to numerous other studies in the field of natural products. This project is relevant to the mission of NCCIH, which is to define the usefulness and role of complementary and integrative health interventions in improving health and health care. Overall, we aim to identify natural product inhibitors of hyaluronidase that can serve as a lead for the development of new anti-virulence treatments for bacterial infections, and to improve upon methods that are applicable to the study of bioactive mixtures used in complementary and integrative health.